JP5751547B2 - Method for forming spherical colonies - Google Patents

Description

  The present invention relates to a method for forming spherical colonies, and particularly relates to an improvement in the formation efficiency and selective culture of spherical colony-forming cells.

  In recent years, research on regenerative medicine has been actively conducted, but regarding skin cells, clinical application of cultured cells has been performed faster than other tissues. In particular, fibroblasts that produce collagen in the dermis are highly proliferative, and the cultured cells can be used as scaffolds for creating transplanted epidermis and as medical materials for artificial dermis, as well as injecting cultured cells directly into the living body. Skin tissue repair and reconstruction have been carried out.

  For the culture of the fibroblasts, adhesion culture is usually employed by utilizing the property of fibroblasts that adhere to and spread in a culture vessel to form a cell layer. Such a culture form is convenient when the cultured fibroblasts are used in a single layer (sheet) state, but when the cells are injected into a living body, the adhered cells are detached from the container, It is necessary to suspend this in a culture solution to obtain a cell suspension suitable for injection treatment. However, the isolated fibroblasts that lost their scaffolds not only survived for a long time in a floating state, but also hardly proliferated even after transplantation, and the engraftment rate after transplantation was not satisfactory.

  By the way, in recent years, it has been reported that a cell population having multipotency exists in the dermis (Non-patent Document 1). This group called skin-derived stem cells / progenitor cells (SKPs) is a kind of somatic stem cells that can be collected from adults as well as various types of skin, nerves, bones, cartilage, fats, etc. It has been shown that it can differentiate into cells. However, the frequency of skin-derived stem / progenitor cells is very low, and particularly for adults, it has been difficult to efficiently culture them. Therefore, if skin-derived stem / progenitor cells can be efficiently cultured, it is expected to be a useful cell source for various regenerative medicine.

Unlike fibroblasts, the skin-derived stem / progenitor cells have the property of proliferating while forming cell aggregates called sphere colonies in a state of floating in a serum-free medium (Non-patent Document) 1). It is known that this spherical colony has a structure in which the cells are integrated with each other than the isolated cells described above, and each cell in the colony maintains a high proliferation power.
It has been attempted to separate fibroblasts and tissue stem cells using the difference in these properties. That is, it is considered that a cell group having a high tissue stem cell concentration can be obtained by adhering fibroblasts to an incubator while performing suspension culture and collecting the formed spherical colonies. However, since tissue stem cells are very rare, the number of tissue stem cells recovered in this way is extremely small, and in order to use them in an isolated state, the supply amount originally corresponds to the demand for regenerative medicine. There is a problem that it cannot be cut.
In addition, when such skin-derived stem / progenitor cells are distributed in the skin tissue, it is considered that they can differentiate into skin cells and become the source of the cells. Is required for infusion therapy, etc., it requires technology to control its pluripotency.

Toma JG, Akhavan M, Fernandes KJ, Barnabe-Heider F, Sadikot A, Kaplan DR, Miller FD, Nat Cell Biol., 3: 778-784, 2001

Considering the properties and problems of each cell described above, fibroblasts that have already been established for application to regenerative medicine in the skin and that are easily available and have a high ability to grow by culturing, such as the above-mentioned skin-derived stem / progenitor cells, Spherical colonization suitable for clinical use such as transportation, injection operation, cell growth source, etc. is considered ideal. Furthermore, although it is also required to create such a spherical colony according to a desired purpose, such a method has not yet been realized.
This invention is made | formed in view of the said problem, and it aims at providing the method of forming the spherical colony of a cell efficiently.

As a result of intensive studies conducted by the present inventors in order to solve the above problems, it has been found that spherical colonies suitable for clinical use can be formed with high efficiency by sequentially culturing target cells under specific conditions. It was. Furthermore, by adjusting the culture conditions, it was found that cell types, colony sizes, numbers, and adherent spherical colonies according to the desired purpose can be created, and the present invention has been completed.
That is, the method for producing a spherical colony according to the present invention includes the following steps (A) to (B).
(A) a step of isolating the target cell;
(B) Rotating / floating culture of the target cell after the step (A).

Moreover, in the said manufacturing method, it is suitable to adhere | attach culture | cultivate further the isolated object cell in the said (A) process.
Moreover, in the said manufacturing method, it is suitable to adjust the cell seeding density in a suspension culture in the range of 1 * 10 < ³ > -1 * 10 < ⁶ > in the said (B) process.
Moreover, in the said manufacturing method, it is suitable in the said (B) process to adjust a floating culture period in the range of 1-3 days.
Moreover, in the said manufacturing method, it is suitable in the said (B) process that the rotational speed in suspension culture is 1-3 rpm.

In the production method, the target cell is preferably an epithelial cell and / or a mesenchymal cell.
Moreover, in the said manufacturing method, it is suitable that the said object cell is a fibroblast and / or a bone marrow stromal cell.
Moreover, in the said manufacturing method, it is suitable that at least one part of the said spherical colony contains a tissue stem / progenitor cell.
Moreover, in the said manufacturing method, it is suitable to perform suspension operation in suspension culture in the said (B) process.
Furthermore, the spherical colony for regenerative medicine / cell therapy according to the present invention is characterized by the production method described above.

  According to the method of the present invention, spherical colonies of cells can be efficiently formed. Since the spherical colony is excellent as a cell transport, injection operation, and cell growth source in clinical use, it can be expected to be used for regenerative medicine and cell therapy. In addition, according to the present invention, it is possible to form efficient spherical colonies for stem / progenitor cells that form spherical colonies but are less frequent, including cells that are usually difficult to form spherical colonies. It is possible to obtain a spherical colony having a size, ease of adhesion, and purity according to the purpose, and thus high contribution to the regenerative medicine / cell therapy is desired.

It is the figure which showed the difference between a spherical colony (sphere colony) and a cell aggregate (cell aggregate). The arrow in FIG. 1a indicates a spherical colony, and the arrow in FIG. 1b indicates a cell aggregate. It is the graph which showed the formation number (FIG. 2a) of the spherical colony and cell aggregate in an isolated cell and a cultured cell, and the average (FIG. 2b) of those magnitude | sizes. It is a figure which shows the spherical colony of a skin origin stem / progenitor cell (Skin-derived precursor cells: SKPs). It is the graph which showed the formation number (FIG. 3a) of the spherical colony and cell aggregate by cell seeding density (1-8 * 10 < ⁵ >), and the average (FIG. 3b) of those magnitude | sizes. It is the graph which showed the formation number (FIG. 4a) of the spherical colony and cell aggregate by a suspension culture period (1-3 days), and the average of those magnitude | sizes (FIG. 4b). “With 3 days” in FIG. 3 indicates a sample cultured for 3 days while performing a suspension operation in suspension culture. It is the graph which showed the formation number (FIG. 5a) and the average (FIG. 5b) of the spherical colony and the cell aggregate by the kind of culture medium (serum-free medium, serum medium) in the floating culture after adhesion culture. It is the fluorescence micrograph of the three-dimensional gel culture of the cell by a spherical colony origin cell and an adhesion | attachment culture | cultivation on the 10th day. It is a fluorescence micrograph of a tissue section two weeks after nude mice transplanted with spherical colony-derived cells and cells by adhesion culture.

Hereinafter, preferred embodiments of the present invention will be described.
The technical difficulty in producing spherical colonies from cultured cells is that the cultured cells are extremely difficult to form spherical colonies. In the first place, the formation of spherical colonies of cells mostly occurred by chance, and there was no technique for intentionally controlling the formation of spherical colonies from cultured cells. Therefore, even if spherical colonies were observed during the culture, the frequency was very small.
Second, spherical colonies are usually characterized in that cells are continuously arranged to form a single structure, but if the conditions are not appropriate, cell aggregates (cells) to which the cells have simply adhered. a state called “aggregate). When further culturing in this state, other spherical colonies or cell aggregates are gradually joined to the aggregates to form a large cell mass. Then, sufficient oxygen and nutrients are not supplied to the central part, and in many cases, these cells are necrotized.
In addition, since spherical colonies are usually mixed in isolated cells floating in the isolated state and the cell aggregates, it is difficult to obtain only spherical colonies from these cells.
According to the present invention, by carrying out cell culture according to a specific process, a spherical colony suitable for cell transplantation treatment such as injection can be obtained without forming the cell mass and removing isolated cells as much as possible. It is to be obtained with high frequency and high purity.
Depending on the treatment, a therapeutic effect by the isolated cells may be expected. Accordingly, the present invention also contemplates obtaining spherical colonies with the isolated cells mixed at an appropriate ratio and using the cells in that state.

  In the present application, the “spherical colony” means a group of proliferated cells forming an independent spherical group in the medium. In general, the term spherical colony is particularly known to refer to an aggregate of stem cells and progenitor cells, but the formation of cell aggregates according to the present application is not limited to stem cells and progenitor cells, In the present application, the term is used for all cells.

The size of the spherical colony in the present invention depends on the cell type to be used. For example, in the case of mammalian fibroblasts, spherical colonies having an average diameter of about 50 to 200 μm are transplanted cell sources for clinical use. Suitable for. In general, if the spherical colony is too small, the proliferation efficiency of the cells based on the colony may be too low, and if the spherical colony is too large, the central cell may be necrotic.
However, conditions such as the size of the spherical colony, the number of cells per colony, the ratio of the spherical colony to the isolated cell, the cell type forming the colony, etc. vary depending on the tissue into which the spherical colony is transplanted and the cell type used, It is preferable to determine according to the purpose. This can be performed by adjusting conditions such as cell density, rotation / floating culture period, medium, etc. in the method according to the present invention described later. That is, the method for producing a spherical colony according to the present invention includes a selective culture method for spherical colony-forming cells (SFCs).

The method for producing a spherical colony according to the present invention includes:
(A) isolating the target cell;
(B) After the step (A), the method includes a step of suspension culture while rotating the target cell.
First, the steps (A) and (B) will be described.

(A) Process In the manufacturing method of the spherical colony concerning this invention, the (A) process represents isolating a target cell.
The type of target cells used in the present invention is not limited, and in particular, epithelial cells including epidermal cells, fibroblasts, bone marrow stromal cells, osteoblasts, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc. The mesenchymal cells are preferably, and more preferably fibroblasts and bone marrow stromal cells having high adhesion. These target cells may be used alone or in combination of two or more. The target cells naturally include stem cells and progenitor cells distributed in each tissue, but the presence of stem cells and progenitor cells is not essential in the spherical colony of the present invention. That is, for example, when obtaining a spherical colony by the method of the present invention from cells (group) obtained from the dermal tissue of the skin, even if all of the obtained colonies are formed from fibroblasts, All may contain stem cells and progenitor cells. Examples of stem cells and progenitor cells that can be included in the spherical colonies of the present invention include skin-derived stem / progenitor cells (SKPs). If at least a part of the spherical colony contains SKPs, the proliferation of cells based on the colony may be enhanced.
In addition, as a collection source of the target cells used in the present invention, for example, mammals such as humans are suitable, but other organisms may be used. Moreover, you may use the cell line derived from the said collection source.

Moreover, the process which concerns on isolation of an object cell can be performed by a well-known method. For example, the tissue is separated into individual cells by appropriately washing and disinfecting a tissue section containing the target cell and treating it in a solution containing a proteolytic enzyme. Examples of proteolytic enzymes include collagenase, dispase, hyaluronidase, elastase, trypsin, pronase and the like. For example, in the case of skin tissue, collagenase and dispase are suitable. A chelating agent such as EDTA may be used for cell detachment.
The isolated cells are washed or separated by a cell strainer as necessary, and are subjected to step (B).

In the step (A), it is preferable to perform adhesion culture (primary culture) using the isolated cells as primary culture cells. Although it is possible to obtain spherical colonies without performing adhesion culture after isolation, the formation rate of spherical colonies can be obtained by performing planar culture (adhesion culture) before floating culture in step (B). Can be further improved.
The adhesion culture is performed by seeding the isolated primary cultured cells on a culture vessel and culturing them in a culture solution or a medium.
When the isolated cells are seeded in a culture container, they adhere to the surface of the container and proliferate while spreading. Although the shape of adhesion / extension varies depending on the cell type, a cell monolayer is usually formed on the surface of the culture vessel by continuing the adhesion culture.
The culture vessel used for the adhesion culture is not particularly limited as long as it is made of a material and shape to which cells can adhere, but generally, glass or plastic 6-well, 12-well, 48-well, 96-well, etc. And culture dishes such as petri dishes and flasks. Collagen or the like may be used as a cell adhesion substrate.

As the culture solution or medium, a basal medium containing inorganic salts, various amino acids, sugars, vitamins and the like can be used. As the basal medium generally used for cell culture, for example, Iscove modified Dulbecco medium (IMDM), RPMI, DMEM, Fischer medium, α medium, Leibovitz medium, L-15, NCTC, F-10, F-12, MEM McCoy medium and the like.
In addition, the culture medium or medium includes the patient's own serum (autologous serum), fetal calf serum (FBS (FCS)), newborn calf serum (NCS), calf serum (CS), horse serum (HS), etc. It is preferable to add serum or a substitute substance thereof. In addition, it is preferable to replace the culture medium and medium during the culture as necessary.

The culture procedure and conditions for each cell type to be used may follow a known cell culture method. For example, in the case of mammalian cells, in addition to setting the culture environment to about 37 ° C. according to body temperature, the cells are cultured in a 5 to 10% CO ₂ atmosphere if necessary for pH adjustment of the medium.
The culture period can be appropriately set according to the cell type, culture container, culture conditions, etc. used, and the standard of culture is until the cell density on the surface of the culture container increases and becomes approximately 80-90% confluent. And
Adherent cultured cells that have been cultured for a set period of time are treated with a proteolytic enzyme such as trypsin or a chelating agent, and detached from the culture vessel. The detached cells are washed or separated by a cell strainer as necessary, and are subjected to step (B).

In the present invention, the adhesion culture may include a process of subcultured cultured cells.
When the primary culture is subcultured, the culture medium is changed and reseeded before the primary cultured cells reach complete confluence.
The passage method is not particularly limited. For example, the culture medium is removed from the primary cultured cells adhered on the culture container, and the cells are washed if necessary, and then a protease such as trypsin or a chelating agent is added to the culture container. Cells that adhere to the cell are detached, recovered, and seeded again in a cell culture container to obtain cells for the first passage. The number of passage cells to be seeded may be determined by appropriately confirming the primary cultured cells. According to a known method, the enzyme reaction with the serum medium is stopped or the cells are washed by centrifugation or the like. Also good.

  The subcultured cells are cultured until approximately 80 to 90% confluent under the same methods and conditions as in the aforementioned adhesion culture. The first-passage cells cultured to the above state are detached from the culture vessel by treatment with the above-mentioned proteolytic enzyme, etc., washed or separated with a cell strainer as necessary, and subjected to step (B). Although the spherical colony concerning this invention can be obtained even if it does not perform the said subculture, after performing a subculture more than twice for the purpose of purifying a target cell more (removing an unnecessary cell) (B). You may proceed to the process. However, considering cell senescence associated with passage, it is generally preferable that passage is performed up to about 3 times.

(B) Process In the method concerning this invention, the (B) process represents carrying out suspension culture, rotating the object cell obtained at the said (A) process.
The target cells isolated in the step (A) are suspended in a culture solution or medium, seeded in a culture container, and suspension culture is started.
Floating culture is a culture method that is performed while rotating or stirring a culture vessel, whereby cultured cells can be grown in a state of floating in a culture solution or medium without adhering to the surface of the vessel. In the present invention, it is particularly preferable to culture while rotating. The target cells seeded in the culture vessel are rotated so that the cells remain floating in the isolated state, and the cells that proliferate uniformly in all directions in the floating state to form spherical aggregates, that is, spherical colonies And divided.

The culture vessel used for the suspension culture in the step (B) is not limited as long as it is suitable for suspension culture, and examples thereof include flasks and tubes, petri dishes and plates for suspension culture, and the like. In the present invention, it is particularly preferable to use a suspension culture tube having a capacity of 14 mL because of ease of handling in setting the cell seeding density described below.
Although the cell seeding density in the suspension culture depends on the type and size of the cells used, spherical colonies can be suitably obtained by setting the cell seeding density in the range of 1 × 10 ³ to 1 × 10 ⁶ cells / mL. For example, in the case of fibroblasts, the formation efficiency of spherical colonies can be further improved by setting the cell seeding density to 3.5 to 7.0 × 10 ⁴ cells / mL.
When the density range is exceeded, the number of spherical colonies formed is somewhat reduced, but the average size of the colonies tends to be larger. Also, when the density is less than the above, the number of spherical colonies formed is reduced, but smaller size colonies may be obtained. That is, in the present invention, a desired spherical colony can be obtained by adjusting the cell seeding density.
The culture conditions (temperature, atmosphere, etc.) of this step can be set according to the above-mentioned adhesion culture or known culture conditions, but the cells in culture are 1 to 3 rpm, particularly 1.3. It is preferable to float at a rotational speed of ˜2 rpm. If the rotational speed is too fast, the efficiency of spherical colony formation may be reduced, and if it is too slow, cell aggregates tend to be formed rather than spherical colonies.

In this step, the size, adhesion, and purity of the spherical colonies can be controlled by adjusting the suspension culture period. For example, when it is necessary to selectively cultivate spherical colonies, the colonies can be obtained with high purity by carrying out suspension culture for 3 days or more. At a stage where the culture period is less than 3 days, a considerable number of isolated cells remain.
Furthermore, it is preferable to perform a suspension operation intermittently during the culture to separate the cell aggregates. In the culture over 3 days or more, the formation of cell aggregates like cell aggregates is confirmed during the culture. Therefore, by performing a suspension operation during culture, the cell mass can be prevented from aggregating and a larger number of colonies can be obtained. The suspension operation is preferably performed by pipetting.

When the suspension culture period is less than 3 days, a mixture of isolated cells and spherical colonies is obtained. Also, spherical colonies can be obtained in an immature state. For example, in suspension culture for about one day, spherical colonies are observed that are mixed with a large number of floating isolated cells, loosely connected between cells, and retain high adhesion ability. By continuing the culture of this spherical colony, it gradually changes into a spherical colony that is difficult to adhere. When this is cultured for 3 days or more, the formation of spherical colonies proceeds, and the number of isolated cells decreases accordingly. Therefore, mature spherical colonies can be selectively recovered.
As described above, spherical colonies of less than 3 days are undeveloped, small in size, and easy to adhere, which is advantageous for some cell transplantation treatments that need to be engrafted immediately after transplantation. On the other hand, spherical colonies that have been subjected to suspension culture for 3 days or more are difficult to adhere and easily maintain a floating state, and thus take time to engraft, but are advantageous for transportation and injection into a living body. Thus, in the present invention, a desired spherical colony can be obtained by adjusting the suspension culture period. Therefore, in the present invention, it is preferable to adjust the suspension culture period to 1 to 3 days according to the purpose.

The culture solution or medium used for suspension culture is not particularly limited as long as it is usually used for suspension culture of cells. In the present invention, it is preferable to use a serum-containing medium and a serum-free medium properly according to the purpose. . When a serum-free medium is used, the number of spherical colonies formed increases, and the size of each spherical colony tends to be slightly smaller. In the case of a serum-containing medium, the number of spherical colonies formed is slightly reduced as compared with a serum-free medium, but it becomes possible to efficiently obtain colonies having a relatively large size. That is, in the present invention, in addition to the cell density, the number and size of spherical colonies can be controlled by adjusting the serum of the medium in suspension culture. In addition, the spherical colony by a serum-free culture medium is very small, and a colony smaller than the size of the spherical colony which can be adjusted by the change of the cell density can be obtained.
The serum here refers to cells such as the patient's own serum (autologous serum), fetal bovine serum (FBS (FCS)), newborn calf serum (NCS), calf serum (CS), horse serum (HS) and the like. Serum normally used for culture, or an alternative substance thereof is shown, and the concentration is adjusted appropriately for use.

  The spherical colony according to the present invention obtained by suspension culture can survive in a suspended state for a long period of time compared to cells cultured normally. That is, normally, cells are gradually lost during transport and injection after the cells are detached, but the spherical colonies according to the present invention are particularly suitable for treatments involving injection into the body for the reasons described above. Furthermore, since the engraftment rate and survival rate after injection are greatly improved, it is suitable for use in, for example, current cell transplantation treatment and regenerative medicine for skin and bone marrow.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, these examples do not limit the present invention.
First, the cell culture method performed in this example is shown in the following (1) to (2).
(1) Primary cell culture (adhesion culture)
A BALB / cAJcl mouse (7 weeks old, male, Nippon Claire) was sacrificed by overdose of somnopentyl (manufactured by Kyoritsu Pharmaceutical Co., Ltd.) intraperitoneally, and then hair was cut with a clipper. The back skin tissue was carefully excised so that the subcutaneous tissue such as fat and blood vessels was not mixed, and the collected tissue (about 1 cm) was immersed in a 12% isodine solution (manufactured by Meiji Seika Co., Ltd.) for 90 seconds for disinfection. . After washing with PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.), it was cut into small pieces of about 1 to 2 mm in a clean bench. This tissue piece was incubated with a collagenase solution (manufactured by Wako Pure Chemical Industries, Ltd.) having a concentration of 10 mg / mL for 1 hour at 37 ° C. and 5% CO _{2 to} carry out enzyme digestion. Next, the operation of centrifuging at 4 ° C. and 1500 rpm for 5 minutes, removing the supernatant, and washing with 5 mL of α-MEM medium (manufactured by Wako Pure Chemical Industries, Ltd.) was repeated twice. Thereafter, the supernatant is removed, and 6 mL of serum-containing α-MEM (α-MEM medium + 10% FBS + penicillin / streptomycin / fungizone (GIBCO)) is added to suspend the cells, and the cell suspension is added to a 6-well plate at 1 mL. Seeded per well. After cell seeding, 1 mL of serum-added α-MEM was further added to each well, followed by culturing at 37 ° C. and 5% CO ₂ . The medium was changed twice a week. Passage was performed using trypsin EDTA (GIBCO) or TrypLE Select (GIBCO) when the cells reached 80-90% confluence. When it was confirmed that the cells had peeled off with a visual microscope, the reaction was stopped with α-MEM containing serum, centrifuged at 4 ° C. and 1500 rpm for 5 minutes, and then the supernatant was removed and resuspended in PBS. Count the number of cells using a hemocytometer, re-centrifuge, suspend in serum-containing medium, re-inoculate the plate or dish with the number of cells according to the purpose, and culture at 37 ° C. and 5% CO ₂ . Continued.

(2) Rotating / floating culture after adhesion culture Cells after adhesion culture in serum-containing α-MEM (that is, after the operation of (1) above) were collected with trypsin EDTA, and centrifuged at 1500 ° C. for 5 minutes. . After washing, the cells were passed through a 70 μm cell strainer, the number of cells was counted, and serum-free medium for SKPs (D-MEM / F12 + 2% B-27 + 20 ng / mLbFGF + 20 ng / mLEGF + penicillin / streptomycin / fungizone) or α-MEM containing serum The cells were resuspended. Inoculate a 14 mL tube (Falcon) with a certain number of cells, and perform rotator (manufactured by BIO CRAFFT) (1.3 rpm) for 1 to 3 days at 37 ° C. and 5% CO ₂ to form spherical colonies. It was.

  After the start of suspension culture in (2) above, some of the cells grew to form spherical colonies, and some of them aggregated to form cell aggregates. In the present application, the case where the cell mass has a spherical structure as shown in FIG. 1a is a “spherical colony” (“sphere colony” in the figure), and a plurality of spherical colonies or cells as shown in FIG. The structure that has become a large cell mass is called “cell aggregate” (“cell aggregate” in the figure). Spherical colonies that take a certain structure and form do not grow indefinitely and the size is kept constant. On the other hand, cell aggregates coalesce and become large during culture, and the morphology is not constant and the size may increase without limitation. Therefore, it was considered that the cell aggregate was not a functional cell structure or unit, but a state where cells and spherical colonies were simply adhered to each other. In addition, cell aggregates become enormous by repeated fusion, and sufficient oxygen and nutrients are not supplied to the inside, so that cells in the center may be necrotized. Therefore, in the present application, the following tests examined the conditions in which spherical colonies as shown in FIG. 1a were efficiently formed and cell aggregates as shown in FIG. 1b were not formed as much as possible.

Spherical colony formation efficiency of isolated cells and cultured cells The isolated cells obtained by the operation of (1) above are obtained by the sample (isolated cells) subjected to rotation / floating culture as it is and the operation of (1) above. The phase-contrast microscope images of the sample (cultured cells) obtained by adhesion culture of the isolated cells and rotation / floating culture were recorded and observed. The number of spherical colonies and cell aggregates in each sample is shown in FIG. 2a, and the average size is shown in FIG. 2b.
As shown by FIGS. 2a and 2b, no apparent spherical colony formation was observed in the isolated cells, but numerous spherical colony formations were observed in the cultured cells.
As a result of further detailed tests, when cells obtained from adult skin tissue as described above were used, the frequency of spherical colonies was extremely low, but depending on the type and state of the cells used, single cells from the tissue were used. It was found that spherical colonies were formed even from detached cells.
In addition, for example, in skin-derived stem / progenitor cells (SKPs), a spherical colony is formed without undergoing adhesion culture or rotation culture (FIG. 3). Since it is particularly low, it is difficult to efficiently obtain the colonies.
In contrast, in the present invention, spherical colonies can be obtained by performing suspension culture while rotating the isolated cells. In particular, it is preferable that the cultured cultured cells that do not normally proliferate in suspension are subjected to suspension culture while being rotated after adhesion culture.

Cell seeding density and spherical colony formation efficiency Cells subjected to adhesion culture by the above operation (1) were collected with trypsin EDTA, and the number of cells was counted. After resuspension in serum-containing α-MEM, put in 14 mL tubes with different cell numbers (1, 2, 4, 8 × 10 ⁵ cells / 8.5 mL), and rotate for 1 day at 37 ° C. and 5% CO _2. Rotational culture (floating culture) at 3 rpm). The number of spherical colonies and cell aggregates formed by each seeding density is shown in FIG. 4a, and the average size is shown in FIG. 4b. The cell count was performed by the following method.

<Measurement of cell number>
100 μL was taken from the cell suspension and mixed with the same amount of trypan blue. 10 μL of this was taken, and the number of cells at 4 locations was counted with a hemocytometer under a phase contrast microscope. The same measurement was performed twice, and the number of cells was calculated from the average value.

As shown in FIGS. 4a and b, the seeding density of the cells clearly affected the number and size of the spherical colonies. In particular, when the number of cells at the start of suspension culture is 4 × 10 ⁵ /8.5 mL (that is, the cell seeding density is 4.7 × 10 ⁴ cells / mL), the number of appropriately sized spherical colonies is large. Been formed. As a result of further tests, in the case of skin cells (fibroblasts) used in this test, spherical colonies are formed particularly efficiently when the cell seeding density is 3.5 to 7.0 × 10 ⁴ cells / mL. It was done.
As a result of further detailed investigation, it was found that spherical colonies can be suitably obtained in the present invention if it is 1 × 10 ³ to 1 × 10 ⁶ cells / mL.

Pipetting and spherical colony formation efficiency Cells subjected to adhesion culture by the above operation (1) were collected with trypsin EDTA, and the number of cells was counted. After resuspension with α-MEM containing serum, the cells were seeded at a cell seeding density of 4.7 × 10 ⁴ cells / mL in a 14 mL tube, and rotator (1.3 rpm) at 37 ° C., 5% CO ₂ for 1, 2, 3 days. Rotational culture (floating culture) was performed. In the case of culturing for 1 to 3 days, and when culturing with pipetting for 3 days (indicated as “3 days present”), spherical colonies and cell aggregates (cell aggregate) ) Is shown in FIG. 5a, and the average size is shown in FIG. 5b.
As shown in FIGS. 5a and 5b, when pipetting was not performed, there were problems such as a small number of spherical colonies formed or many cell aggregates. On the other hand, in the sample cultured while pipetting, spherical colonies increased remarkably and there were few cell aggregates.
Therefore, in the present invention, it is preferable to perform suspension culture for 3 days or more after adhesion culture. Furthermore, it is more preferable to suspend the cells in suspension culture by pipetting.

Type of medium at the time of re-suspension and efficiency of spherical colony formation The same number of cells subjected to adhesion culture by the operation of (1) above was used as serum-free medium for SKPs (D-MEM / F12 + 2% B-27 + 20 ng / mLbFGF + 20 ng / mLEGF + penicillin / streptomycin) / Fungizone) or resuspended in serum-containing α-MEM, placed in a 14 mL tube, and subjected to rotary culture (floating culture) with a rotator (1.3 rpm) at 37 ° C. and 5% CO ₂ for 3 days (cell seeding) Density: 4.7 × 10 ⁴ cells / mL). Fig. 6a shows the number of spherical colonies and cell aggregates formed in suspension culture in serum-free medium and in suspension culture in serum medium. The average is shown in FIG.
As shown in FIGS. 6a and b, when cultured in serum-free medium, the number of spherical colonies tends to be large and the size tends to be small. When cultured in serum-containing α-MEM, the number of spherical colonies is small and the size increases. There was a trend.
Therefore, it was suggested that the number and size of spherical colonies to be formed can be adjusted in the rotation / floating culture after the adhesion culture by selecting the medium. That is, in the present invention, it is possible to create a spherical colony corresponding to a desired treatment by adjusting the medium.

Properties of cells cultured with spherical colony-derived cells The cultured cells obtained by the above operation (1) were fluorescently stained with the following PKH26, and suspended colony was performed for 3 days by the above operation (2) to obtain spherical colonies. . Subsequently, the collected spherical colonies were three-dimensionally cultured with the following gel. This gel culture is widely used as a model of skin connective tissue, and is used this time as a cell transplant to connective tissue.
In addition, the adherent cultured cells obtained by the above operation (1) were fluorescently stained with the following PKH26 and suspended (no suspension culture) was similarly subjected to three-dimensional gel culture.
Among samples using spherical colonies, fluorescence micrographs on the 10th day of each three-dimensional gel culture for samples obtained by suspension culture for colony formation in 10% FBS medium and samples obtained in serum-free medium. Is shown in FIGS. 7A and 7B. Further, FIG. 7C shows a fluorescence micrograph of a sample (derived from adherent cultured cells) on day 10 of three-dimensional gel culture in which the cultured cells of (1) are suspended without suspension culture. The photographs in FIGS. 7A to 7C are the same samples taken in 3 to 4 fields of view.
The fluorescent staining with PKH and the Matrigel culture were performed by the following method.

<Fluorescent staining with PKH26>
The cultured cells (2 × 10 ⁷ cells) obtained by the above operation (1) were lightly centrifuged and washed twice with PBS. After centrifugation at 400 × g for 5 minutes, the supernatant was aspirated so that no more than 25 μL of supernatant remained in the cell pellet. Thereafter, the cell suspension obtained by adding 1 mL of Diluent C to the cell pellet and mixing was immediately stained.
1 mL of the cell suspension and 1 mL of the 2 × stain solution (4 μL of PKH26 (4 × 10 ⁻⁶ M dye solution obtained by adding (No. P9691) to 1 mL of Diluent C)) are rapidly suspended by pipetting. Turbid and incubated for 2-5 minutes at 25 ° C. To stop the reaction, an equal volume (2 mL) of serum or protein solution was added to the reaction and incubated for 1 minute.
After centrifugation at 400 × g and 25 ° C. for 10 minutes, the supernatant was removed, and 10 mL of PBS was added and transferred to a new tube. Subsequently, centrifugation was performed at 400 × g and 25 ° C. for 5 minutes, the supernatant was removed, and 10 mL of PBS was added twice or more to wash the cells.

<3D gel culture>
Matrigel was dissolved at 4 ° C., and all operations were performed on ice.
For cells in adhesion culture, 2.0 × 10 ⁵ cells were seeded on each well and suspended in 112.5 μL of medium. On the other hand, the spherical colony obtained by the above operation (2) was centrifuged together with the medium, the supernatant was removed, and the suspension was similarly suspended in 112.5 μL of the medium. In addition, the cell number at the time of producing a spherical colony was made to correspond with the cell number (2.0 * 10 < ⁵ >) at the time of suspending adhesion culture.
Using a round tube, add 4 μL of sterilized 1N NaOH to 182.25 μL of collagen type 1 to adjust the pH (turn red), add 20.25 μL of M199 (× 10) to neutralize the collagen. 135 μL of growth factor-reduced Matrigel was suspended, and the cell suspension was further mixed (this operation is performed quickly so that Matrigel does not harden). Thereafter, 150 μL each was placed on a 48-well dish and cultured in a 5% CO ₂ incubator.

In the sample obtained by rotator culture in 10% FBS medium to form a spherical colony, the cells around the colony had already started to proliferate on the third day of culture and spread to the periphery at the stage of the sixth day of culture (FIG. 7A). . In the sample using spherical colonies cultured in a serum-free medium in a rotator, it was confirmed that surrounding cells spread in the gel although the colonies were small (FIG. 7B).
On the other hand, cells cultured in a dish and isolated (detached) did not show much growth in the gel. Furthermore, a slight decrease in the number of cells was observed as the culture was performed (FIG. 7C). In other words, cells that were isolated after adhesion culture as in the prior art did not provide a sufficient effect as a cell source.

In order to further confirm the above results, spherical colonies with serum-containing medium and serum-free medium, which were prepared by the above-described method and stained with PKH26, were injected subcutaneously into nude mice and taken out after 2 weeks. Adherent culture-derived cells not subjected to suspension culture were also transplanted in the same manner after staining with PKH26 immediately before transplantation. The number of transplanted cells was 1 × 10 ⁶ for isolated cells derived from adhesion culture, and 4 × 10 ⁵ at the start of culture for spherical colonies. The excised tissues were frozen sections and each was observed under a fluorescence microscope. A micrograph of each sample is shown in FIG.
As shown in FIG. 8, when a spherical colony obtained by floating culture in serum-containing or serum-free medium is used, the cells proliferate from the colony to the surroundings, while the colony remains in the shape of the colony. However, the number of engrafted cells was large. On the other hand, even when cells derived from adhesion culture were used, the cells were engrafted but scattered in the subcutaneous connective tissue, and the engraftment rate was not high.
From the above results, the spherical colony-derived cells of the present invention are useful for cell transplantation treatment.

Claims (4)

The manufacturing method of the spherical colony characterized by including the following (A)-(B) process.
(A) a step of adhesion culture to isolate fibroblasts,
(B) After the step (A), the fibroblasts are separated from the culture vessel and separated , adjusted so that the cell seeding density is 1 × 10 ³ to 1 × 10 ⁶ cells / ml, and 1 to ³ rpm. The process of rotating and floating culture at a rotational speed of . In the said (B) process, a floating culture period is adjusted in the range of 1-3 days, The manufacturing method of the spherical colony of Claim 1 characterized by the above-mentioned. The method for producing a spherical colony according to claim 1 or 2 , wherein at least a part of the spherical colony contains a tissue stem / progenitor cell. The method for producing a spherical colony according to any one of claims 1 to 3 , wherein in the step (B), a suspension operation is performed during suspension culture.